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Originally published online as doi:10.1189/jlb.1104644 on March 23, 2005

Published online before print March 23, 2005
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(Journal of Leukocyte Biology. 2005;78:144-157.)
© 2005 by Society for Leukocyte Biology

CTLA4-CD80/CD86 interactions on primary mouse CD4+ T cells integrate signal-strength information to modulate activation with Concanavalin A

Sambuddho Mukherjee, Asma Ahmed and Dipankar Nandi1

Department of Biochemistry, Indian Institute of Science (IISc), Bangalore, India

1 Correspondence: #126, Department of Biochemistry, Indian Institute of Science, Bangalore, 560012 India. E-mail: nandi{at}biochem.iisc.ernet.in


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ABSTRACT
 
The mechanisms by which concanavalin A (Con A), a lectin, activates T cells are poorly studied. A low dose of Con A is stimulatory for T cells, whereas a high dose of Con A results in suppression of proliferation and enhanced T cell death. The expression and functional roles of costimulatory receptors, CD28 and cytotoxic T-lymphocyte antigen 4 (CTLA4), and their ligands, CD80 and CD86, on primary mouse CD4+ T cells after activation with different doses of Con A were studied. CTLA4-CD80/CD86 interactions in this T:T cell activation model demonstrate distinct outcomes depending on the dose of Con A. CTLA4-CD80/CD86 interactions inhibit CD4+ T cell cycling and survival after activation with a suppressive dose of Con A by increasing oxidative stress and decreasing levels of BclXL. The enhanced CD4+ T cell death with a suppressive dose of Con A is dependent on excess H2O2 and nitric oxide but is independent of Fas and caspase activity. It is surprising that the increased proliferation of CD4+ T cells with a suppressive dose of Con A on blocking CTLA4-CD80/CD86 interactions is largely interleukin (IL)-2-independent but is cyclosporine A-sensitive. On activation with a stimulatory dose of Con A, CTLA4-CD80/CD86 interactions enhance T cell activation and survival by reducing the production of reactive oxygen species, increasing IL-2 and BclXL levels. Here IL-10 but not transforming growth factor-ß plays a functional role. In summary, CTLA4-CD80/CD86 interactions on T cells integrate signal strength, based on the dose of Con A, to enhance or inhibit primary mouse CD4+ T cell cycling and survival.

Key Words: costimulation • T cell cycling and survival • IL-2-independent • oxidative stress • IL-10 • TGF-ß


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INTRODUCTION
 
Optimal activation of CD4+ T cells requires two distinct signals: Signal 1 is T cell receptor (TCR)-CD3-mediated, arising from interaction between the TCR and the cognate major histocompatibility complex (MHC)- peptide complex, and signal 2 is antigen-independent and involves the binding of the costimulatory receptor CD28 on T cells to its ligands CD80/CD86 on antigen-presenting cells (APCs). CD28 has a long half-life and is constitutively expressed on the surface of T cells. CD28 binding to CD80/CD86 together with TCR signaling (signal 1) lead to the production of high levels of interleukin (IL)-2 and other cytokines followed by increased T cell cycle progression. In addition, CD28 signaling enhances survival by increasing levels of antiapoptotic proteins, including BclXL. Cytotoxic T-lymphocyte antigen 4 (CTLA4; CD152), which is closely related to CD28, is another important T cell costimulatory receptor. CTLA4 has a short half-life, and the majority of molecules is present intracellularly with low levels of cell-surface expression. CTLA4 binding to CD80/CD86 results in decreased IL-2 production and T cell cycle progression. The opposing roles of CD28 and CTLA4 during T cell activation are reinforced by the phenotype of mice lacking these costimulatory receptors. cd28–/– mice are able to initiate but are unable to sustain T cell immune responses. A more dramatic phenotype is displayed by ctla4–/– mice, which die within 3–4 weeks of age as a result of hyperproliferation of CD4+ T cells (reviewed in refs. [1 2 3 ]).

One mechanism by which CTLA4 dampens T cell activation is by binding to CD80/CD86 with at least tenfold higher affinity and sequestering these ligands from binding to CD28. In addition, the cytoplasmic tail of CTLA4 is required for optimal inhibition of T cell activation [4 , 5 ]. CTLA4 ligation also reduces extracellular signal-regulated kinase activation [6 ], lowers IL-2 production, and reduces T cell cycling [7 , 8 ]. In the initial study, CTLA4 ligation in primary CD4+ T cells, together with anti-CD3 activation, reduced T cell cycling without affecting survival [9 ]. However, a subsequent study using a transgenic TCR mouse demonstrated that CTLA4 expression reduced primary CD4+ T cell cycling and survival [10 ]. The relationship between CTLA4 ligation and survival is controversial, and some reports demonstrate a role in enhancing [11 12 13 14 ] or inhibiting [15 16 17 ] T cell survival. CTLA4 ligation results in T cell anergy in some systems [18 19 20 ] but not others [21 , 22 ]. Also, CTLA4 ligation results in increased levels of transforming growth factor-ß (TGF-ß) [23 , 24 ], but the functional role of this TGF-ß is controversial [25 ]. Although CTLA4 clearly plays important roles in the T cell immune response, the mechanisms by which it acts are not fully understood.

T cells are known to express costimulatory receptors, and their ligands, although the functional consequences of these interactions, are not well studied. B7 molecules on mouse T cells are hypoglycosylated and bind to CTLA4 but not CD28 [26 , 27 ]. In addition, differences in the roles of CD80 on T cells and APCs are known [28 ]. Finally, these interactions may be clinically important, as shown in a disease model [29 ]. In general, CTLA4 has been shown to inhibit T cell responses, although there are some studies that demonstrate a role of CTLA4 in enhancing T cell activation [13 , 30 31 32 33 34 ]. In fact, a recent report demonstrated that a single-chain Fv ligand to CTLA4 enhances T cell activation [34 ]. However, it is unclear whether the inhibiting or enhancing roles of CTLA4 in the literature are a result of the use of different model systems, and one is unable to predict when CTLA4 would act as an enhancer or an inhibitor of CD4+ T cell responses. To study the functional roles of the B7 family of costimulatory receptors and ligands on T cells, we developed a primary CD4+ T cell activation model and demonstrated that CTLA4-CD80/CD86 interactions inhibit or enhance primary CD4+ T cell activation depending on the stimulatory conditions used: Activating CD4+ T cells with plate-bound anti-CD3 and blocking CTLA4-CD80/CD86 interactions increase T cell proliferation; i.e., CTLA4-CD80/CD86 interactions inhibit T cell activation. Conversely, activating CD4+ T cells with phorbol 12-myristate 13-acetate (PMA; P) and ionomycin (I) and blocking CTLA4-CD80/CD86 interactions greatly inhibit T cell proliferation; i.e., CTLA4-CD80/CD86 interactions enhance T cell activation [13 ].

Concanavalin A (Con A) is a lectin that binds to cell-surface glycoproteins, including the TCR, and has been used extensively to study T cell activation [35 36 37 38 39 40 ]. In this report, we studied the functional consequences of blocking CTLA4-CD80/CD86 interactions after activating CD4+ T cells with different amounts of Con A. We show that with a stimulatory dose of Con A, CTLA4-CD80/CD86 interactions enhance, whereas with a suppressive dose of Con A, the same interactions inhibit CD4+ T cell cycling and survival. This study clearly demonstrates that CTLA4-CD80/CD86 interactions integrate signal strength, based on the dose of Con A, to modulate primary mouse CD4+ T cell cycling and survival.


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MATERIALS AND METHODS
 
Mice
CD4+ T cells were obtained from C57BL/6 mice, usually 6- to 10-weeks old. Mice were obtained from the Central Animal Facility [Indian Institute of Science (IISc), Bangalore, India] or the National Institute of Nutrition (Hyderabad, India) and housed in our departmental facility, as per institutional guidelines.

Media, antibodies, and cell lines
Primary CD4+ T cells were cultured in RPMI 1640, supplemented with 25 mM HEPES (Sigma Chemical Co., St. Louis, MO), 2 mM L-glutamine (Life Technologies, Gaithersburg, MD), 5 µM ß-mercaptoethanol (Merck, Rahway, NJ), 100 µg/ml penicillin, 250 µg/ml streptomycin, 50 µg/ml gentamycin (HiMedia Labs, Mumbai, India), and 5% heat-inactivated fetal bovine serum (FBS; Sigma Chemical Co.). Anti-CD3 (145-2C11), anti-CD28 (37.51), and hamster control antibody were sourced from eBioScience (San Diego, CA). Ascites containing anti-CTLA4 and murine CTLA4 human immunoglobulin G1 (mCTLA4hIgG1) were used for all blocking studies, as described previously [13 ]. Anti-CD8 (3.155) and heat-stable antigen (J11D) culture supernatants were used to purify lymph node CD4+ T cells. All other antibodies (e.g., anti-IL-2, anti-IL-4, and others) were obtained from eBioScience. Anti-TGF-ß1 was kindly provided by Dr. Paturu Kondaiah (IISc). For flow cytometry, anti-BclXL was obtained from eBioScience, and secondary antibodies were from Jackson ImmunoResearch Laboratories (West Grove, PA).

Isolation of CD4+ T cells and activation
CD4+ T cells were purified by complement-mediated lysis of J11D+ and CD8+ cells, as described previously [13 ]. Live cells were obtained by density gradient centrifugation with Histopaque 1083 (Sigma Chemical Co.) and subjected to panning over a T25 flask coated with 100 µg/ml anti-mouse Ig (Jackson ImmunoResearch Laboratories). CD4+ T cell preparations were typically ~95% pure, as measured by flow cytometric analysis for key markers. Purified T cells were plated at 6–7 x 104 cells/well in 96-well U-bottom plates (Costar, Corning Inc., NY) in a final volume of 100 µl/well. To minimize nonspecific adhesion of monoclonal antibodies (mAb) to the plate, all wells were precoated with RPMI 1640 containing 5% FBS. In most assays, T cells were activated with different doses of Con A (Sigma Chemical Co.), as mentioned in the figure legends. Anti-CD28 was used at a concentration of 0.3–0.5 µg/ml, and anti-CTLA4 and mCTLA4hIgG ascites were used at a final concentration of 1:100. Fetuin, glutathione (GSH), catalase, N-methyl-L-arginine (L-NMA; all from Sigma Chemical Co.), IL-2, IL-4 (PeproTech, Israel), and cyclosporine A (CsA; Sigma Chemical Co.) were titred and used at the indicated concentrations. Unless otherwise mentioned, CD4+ T cell cultures were pulsed 36 h after activation with ~0.4 µCi/well [3H]-thymidine (BRIT, Mumbai, India) and harvested 12 h later. Incorporated radioactivity was measured using a liquid scintillation counter (Beckman LS6500) to assess levels of proliferation. The data are presented as mean ± SD of replicates in one representative of multiple individual experiments.

Cytokine assays
Supernatants from T cell assays were collected at different time-points or 36 h after activation, and cytokine-specific enzyme-linked immunosorbent assay (ELISA; eBioscience) or bioassays were performed for IL-2 and TGF-ß [using the cell line CCL64], respectively. The amount of cytokine in the supernatants was determined using an equation derived from values obtained from known amounts of standard cytokines, and specific T cell secretion of cytokines was determined by deducting appropriate controls. ELISA was performed with standard amounts of recombinant IL-2 (rIL-2) and various dilutions of culture supernatants. Typically, the linear detection range of the IL-2 assay was 30–900 pg/ml. Active TGF-ß was measured as an index of growth inhibition of CCL-64 cells, which were cultured (~5000 cells/well) with supernatants or with known amounts of rTGF-ß1 (PeproTech), as described previously [13 ]. The linear detection range of the TGF-ß bioassay was 80–1250 pg/ml.

Flow cytometric analysis
For surface staining, ~2 x 105 cells were washed in cold Hanks’ balanced saline solution (Sigma Chemical Co.), containing 0.5% FBS, stained with pretitred amounts of culture supernatants or direct conjugates, washed, and incubated with the appropriate fluorescein isothiocyanate (FITC)-conjugated, preadsorbed secondary antibodies. For intracellular staining of BclXL, cells were fixed with 4% paraformaldehyde (E Merck, San Diego, CA) and permeabilized with 0.2% saponin (Sigma Chemical Co.) prior to staining. Flow cytometry was performed on FACScan (Becton Dickinson, San Jose, CA) using CellQuest (Becton Dickinson) software for acquisition and WinList (Verity, Topsham, ME) software for analysis. Debris and cellular fragments were excluded from the analysis by electronic logical gates based on forward- and side-scatter profiles. Cell-cycle analysis was performed using propidium iodide (PI; Sigma Chemical Co.), as reported previously [13 ]. Production of peroxides,peroxinitrites, and other reactive oxygen species (ROS) was assessed using the oxidation-sensitive fluorescent probe 2',7'-dichlorofluorescein diacetate (DCFDA). Cells treated under the different conditions were incubated with 2.5 µM DCFDA for 20 min and acquired on a FACScan. The total membrane potential was measured on a FACScan by incubating cells with the membrane potential-responsive 3,3'-dihexyloxacarbocyanine iodide (DiOC6; Sigma Chemical Co.), as reported previously [13 ].


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RESULTS
 
Distinct roles of CTLA4-CD80/CD86 interactions during activation of mouse CD4+ T lymphocytes are dependent on the dose of Con A
Purified CD4+ T cells were obtained from mouse lymph nodes and activated with different amounts of Con A [38 ] in the presence of control antibody, anti-CD28, anti-CTLA4, or mCTLA4 (Fig. 1A ). At extremely suboptimal doses (0.2 µg/ml), little proliferation was observed. In keeping with the known role of CD28 in reducing the threshold of primary signal required for activation, T cells activated with a suboptimal dose of Con A and anti-CD28 showed increased proliferation. Con A, at stimulatory doses (e.g., 1 µg/ml), increased T cell proliferation, which was further enhanced with anti-CD28. However, at suppressive doses of Con A (3–4.25 µg/ml), proliferation of CD4+ T cells together with control antibody was suppressed greatly, and triggering with anti-CD28 was not able to rescue proliferation. We used this system to study the roles of CTLA4-CD80/CD86 interactions, using soluble anti-CTLA4, which blocks the interaction of CTLA4 with CD80/CD86, and the monovalent reagent mCTLA4hIgG1, which binds CD80 and CD86 and blocks their interactions with CD28 and CTLA4 [41 ]. Activation of T cells with a stimulatory dose of Con A and soluble anti-CTLA4 or mCTLA4IgG1 decreased proliferation compared with control antibody. However, as Con A concentrations were increased (2.5–4.25 µg/ml), this inhibition of proliferation disappeared gradually, and enhanced proliferation was observed. At suppressive doses of Con A, which are known to enhance T cell death [38 ], blockade of CTLA4-CD80/CD86 interactions enhanced T cell proliferation over and above that in cells treated with control antibody or anti-CD28. Similar results were obtained with a combination of blocking antibodies to CD80 and CD86, which mimicked the effects of mCTLA4hIgG1 (Fig. 1B) . However, this effect was not observed with antibodies against the adhesion molecule CD62L (Fig. 1B) , demonstrating that the effects were specific to CTLA4-CD80/CD86 interactions. These results clearly demonstrated that CTLA4-CD80/CD86 interactions on CD4+ T cells modulated T cell activation depending on the dose of Con A used for T cell activation.



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  		Figure 1. Plasticity of CTLA4-CD80/CD86 interactions on primary CD4+ T cells depends on the dose of Con A used for activation. (A) Primary lymph node mouse CD4+ T cells were isolated and activated with different concentrations of Con A together with control antibody (5 µg/ml), anti-CD28 (aCD28; 0.3 µg/ml), anti-CTLA4 (1:100), or mCTLA4hIgG1 (1:100) for 36 h and pulsed for 12 h with 3H-thymidine. (B) Different amounts of purified antibodies against CD80 and CD86 were added to culture at 0 h and compared with anti-CTLA4 and mCTLA4 (1:100), control antibody (Ctrl Ab), or anti-CD62 ligand (CD62L). (C) The kinetics of T cell activation was studied with a stimulatory (1 µg/ml) or a suppressive (4 µg/ml) dose of Con A together with antibodies for the indicated duration, including the final 12-h pulse with 3H-thymidine. (D) CD4+ T cells were activated with a stimulatory or a suppressive dose of Con A, and control antibody, anti-CD28, anti-CTLA4, or mCTLA4hIgG1 was added at the indicated time-points. Cells were harvested after 48 h of activation. cpm, Counts per minute.

CTLA4-CD80/CD86 interactions are required early during CD4+ T cell activation for manifestation of Con A responses
Next, we studied the activation kinetics of CD4+ T cells stimulated with a stimulatory or a suppressive dose Con A (Fig. 1C) . The optimal proliferation of CD4+ T cells activated with a stimulatory dose of Con A was observed at 36 h after activation. In keeping with the known ability of CD28 to enhance and sustain T cell-proliferative responses, CD4+ T cells activated with a stimulatory dose of Con A and anti-CD28 displayed high, proliferative responses, optimally at 48 h after activation. Similar to Figure 1 , A and B, blockade of CTLA4-CD80/CD86 interactions reduced proliferation in T cells activated with a stimulatory dose of Con A. Also, T cells activated with a suppressive dose of Con A, together with control antibody or anti-CD28, displayed little proliferation. However, blockade of CTLA4-CD80/CD86 interactions greatly enhanced proliferative responses in T cells activated with a suppressive dose of Con A, optimally after 36 h post-activation. To understand the roles of these interactions during CD4+ T cell activation, anti-CD28, anti-CTLA4, or mCTLA4hIgG1 was added at 0, 3, 6, and 12 h of T cell activation with a stimulatory or a suppressive dose of Con A (Fig. 1D) . The modulation of T cell proliferation as a result of CTLA4-CD80/CD86 blockade, with a stimulatory or a suppressive dose of Con A, was most evident when blockade was applied early, i.e., within 6 h of activation. It is possible that a suppressive dose of Con A generated an overly strong, primary signal that reduced T cell proliferation. However, blockade of CTLA4-CD80/CD86 interactions enhanced T cell proliferation in the presence of this strong signal, suggesting that these interactions played an inhibitory role. In keeping with the same logic, the reduced proliferation with blockade of CTLA4-CD80/CD86 interactions with a stimulatory dose of Con A suggests that these interactions enhanced T cell activation under this condition.

Costimulatory receptors and their ligands are expressed on CD4+ T cell activation with Con A
We studied the expression of cell-surface molecules on lymph node primary mouse CD4+ T cells before and after activation with different doses of Con A (Fig. 2 ). Flow cytometric analysis revealed that the vast majority of cells was CD4+ and CD3+. CD28 was constitutively expressed on T cells, and increased levels were detected on activation with a stimulatory or a suppressive dose of Con A. CTLA4 was not detected on unstimulated cells but was induced by 12 h of activation. A similar profile was observed with CD80, which was present at low levels on unactivated T cells. CD86 was also strongly up-regulated upon activation with a stimulatory and a suppressive dose of Con A. Thus, in this T cell:T cell interaction model, costimulatory receptors and ligands are present on CD4+ T cells after activation with Con A.



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  		Figure 2. Primary mouse CD4+ T cells express surface costimulatory receptors and their ligands upon Con A stimulation. Lymph node CD4+ T cells (0 h) were activated with a stimulatory (1 µg/ml) or a suppressive (4 µg/ml) dose of Con A for 12 h or 42 h and stained with specific mAb to different cell-surface markers followed by flow cytometric analysis. The light-gray, dotted lines indicate control antibody; solid gray lines with gray arrows indicate unactivated cells; thin black lines with black arrows indicate 12 h-activated cells; and solid black lines with large arrowheads indicate cells activated for 42 h. Mean fluorescence intensities (MFIs) are indicated in three rows representing 0, 12, and 42 h of activation. This pattern of expression is representative of four independent experiments.

CTLA4-CD80/CD86 interactions modulate levels of ROS, membrane potential, cell-cycle progression, and death, depending on the dose of Con A used for activation
We measured different phenotypic manifestations of activation of primary CD4+ T cells activated with a stimulatory or a suppressive dose of Con A together with different antibodies. Intracellular ROS was detected using the oxidation-sensitive fluorescent probe DCFDA, which results in increased fluorescence upon oxidation. As seen in Figure 3 A , T cells activated with a suppressive dose of Con A displayed over 2.5-fold more intracellular ROS compared with T cells activated with a stimulatory dose of Con A and control antibody. Treatment with anti-CD28 did not significantly change ROS levels with a stimulatory or a suppressive dose of Con A. With a stimulatory dose of Con A, levels of ROS increased on blockade of CTLA4-CD80/CD86 interactions. This profile was reversed with a suppressive dose of Con A, where the blockade reduced ROS to levels observed in T cells activated with a stimulatory dose of Con A and control antibody. The ROS profile observed, under different conditions, is similar at 24 h and 36 h of activation (Fig. 3B) . Next, we used DiOC6 to measure membrane potential changes in these cells; this dye accumulates primarily in mitochondria that maintain mitochondrial membrane potential, thereby showing high fluorescence. T cells treated with a stimulatory dose of Con A and control antibody displayed ~1.5-fold higher fluorescence compared with cells activated with a suppressive dose of Con A (Fig. 3C) . Activation of T cells with a suppressive dose of Con A and anti-CD28 increased membrane potential, but only a slight increase was observed with a stimulatory dose of Con A. CTLA4-CD80/CD86 blockade of T cells slightly reduced membrane potential in cells activated with a stimulatory dose of Con A at 24 h of activation but resulted in over 60% enhancement when activation was carried out with a suppressive dose of Con A. As observed in Figure 3D , significant reduction in membrane potential was observed in T cells activated for 36 h with a stimulatory dose of Con A together with CTAL4-CD80/CD86 blockade. The kinetics of ROS production (Fig. 3B) and membrane potential (Fig. 3D) demonstrates that excess ROS is produced before reduction in membrane potential in T cells activated with a stimulatory dose of Con A together with CTLA4-CD80/CD86 blockade.



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  		Figure 3. Distinct differences in levels of ROS, membrane potential, cell-cycle progression, and hypodiploid population are observed on activation of primary CD4+ T cells with a stimulatory (1 µg/ml) or a suppressive (4 µg/ml) dose of Con A together with blockade of CTLA4-CD80/CD86 interactions. (A) Total levels of ROS were detected using DCFDA and flow cytometric analysis profiles after 24 h of activation are depicted, and numbers in the plot indicate MFI. (B) The relative levels of ROS under different conditions after 24 h or 36 h of activation are represented as mean ± SE of three independent experiments. (C) Membrane potential was studied using DiOC6 fluorescence after 24 h of activation. (D) The relative changes in membrane potential under different conditions at 24 h and 36 h of activation are represented as mean ± SE of three independent experiments. (E) Cell-cycle progression and hypodiploidy were studied using PI staining of CD4+ T cells, activated with a stimulatory or a suppressive dose of Con A together with the indicated antibodies. (F) A kinetic profile of cell cycling and hypodiploidy with mean ± SE of four independent experiments is also depicted for T cells activated with a stimulatory and a suppressive dose of Con A. The results are normalized to cells treated with control antibody alone. The numbers in the plots represent the mean percentage of the cell population in G0/G1, S/G2M, and hypodiploid phases, respectively.

Next, cells in the S/G2M phase of the cell cycle and hypodipoid cells, were detected by PI staining, under different conditions of activation. CD4+ T lymphocytes activated with a suppressive dose of Con A, and control antibody showed a reduced proportion of actively cycling cells and an enhanced hypodiploid population compared with cells activated with a stimulatory dose of Con A after 48 h of activation (Fig. 3E and 3F) , which is consistent with the results obtained with kinetics of proliferation (Fig. 1C) . No major differences were observed on CD28 triggering in conjunction with a suppressive dose of Con A. However, T cells activated with a stimulatory dose of Con A and anti-CD28 demonstrated the highest proportion of actively cycling cells and the lowest proportion of hypodiploid cells (Fig. 3E and 3F) . Conversely, T cells activated with a stimulatory dose of Con A and CTLA4-CD80/CD86 blockade showed a decreased proportion of cycling cells, with a concomitant, ~1.4-fold increase in hypodiploid cells over time compared with the control population. T cells activated with a suppressive dose of Con A and CTLA4-CD80/CD86 blockade demonstrated an ~3.5-fold increase in the actively cycling population, coupled with the reduced hypodiploid population. Thus, an inverse correlation was observed across multiple phenotypic effects on blockade of CTLA4-CD80/CD86 interactions in primary CD4+ T cells activated with stimulatory or suppressive doses of Con A.

Differential expression of BclXL and functional ROS-mediated effects during Con A-mediated activation of primary CD4+ T cells
The decreased proliferation and increased T cell death in CD4+ T cells activated with a suppressive dose of Con A led us to address the roles of Fas, caspase, oxidative stress, and BclXL levels in this culture system [42 , 43 ]. We studied the roles of Fas, using lpr–/– mice, which contain a point mutation in Fas, rendering it nonfunctional. In other studies, the pan-caspase inhibitor, BDfmk, was used to study the roles of caspases. CD4+ T cells were activated for 36 h with P + I, washed, and rested for 36 h as a positive control. Reactivation of these T cells with plate-bound anti-CD3 resulted in 41% of hypodiploid cells in lpr+/–, whereas death was reduced to 23% in lpr–/– T cells (data not shown), clearly demonstrating the role for Fas during activation-induced T cell death (AICD). In the same model of AICD, T cell death in cultures treated with BDfmk was 37% compared with 62% hypodiplody in cultures treated with the control peptide, zFAfmk, demonstrating that caspase activity is required for AICD (data not shown). However, the use of lpr–/– mice or the use of BDfmk failed to modulate proliferation or cell death (data not shown), demonstrating that Fas-Fas ligand (FasL) binding and caspase activity were not involved during Con A-mediated T cell activation (Fig. 4A and 4B ).



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  		Figure 4. ROS-mediated cell death and BclXL expression depend on the dose of Con A used for CD4+ T cell activation. (A) Lymph node CD4+ T cells from Ipr+/– or Ipr–/– mice were activated for 36 h in the presence of a stimulatory (1 µg/ml) or a suppressive (4 µg/ml) dose of Con A, and an index of proliferation was obtained. (B) Cells were treated at 0 time with the pan-specific caspase inhibitor BDfmk or the control peptide zFAfmk. 3H-Thymidine counts are depicted after activation for 36 h with a 12-h pulse. (C) Intracellular staining of the survival factor BclXL was followed by flow cytometric analysis at ~36 h of activation. The numbers in the plots indicate the MFI of the population. (D) T cells were activated with a stimulatory or a suppressive dose of Con A in the absence or presence of different antioxidants for 48 h, and cell-cycle analysis was performed. The antioxidants were used at the following concentrations: GSH, 50 µM; catalase (Cat), 10 µg/ml; L-NMA, 250 µM. The numbers in the plots represent the mean percentage of the cell population in G0/G1, S/G2M, and hypodiploid phases, respectively. (E) The effect of antioxidants in modulating T cell cycling and hypodiploidy under different conditions is represented as mean ± SE of three independent experiments.

Next, we studied the expression of the survival factor BclXL in CD4+ T cells activated under different conditions. There was over threefold reduced expression of BclXL in cells treated with a suppressive dose of Con A and control antibody, as compared with those treated with a stimulatory dose of Con A. CD28 promotes T cell survival by enhancing BclXL levels [44 ]. Although T cells activated with a suppressive dose of Con A and anti-CD28 increased intracellular expression of BclXL (Fig. 4C) , these levels were probably insufficient to enhance T cell cycling and survival on activation with this dose of Con A (Fig. 3E) . CTLA4-CD80/CD86 blockade with a suppressive dose of Con A resulted in an approximate fourfold increase in BclXL levels over the control population. However, with a stimulatory dose of Con A, an ~2.5-fold decrease in BclXL levels was observed on CTLA4-CD80/CD86 blockade. Thus, CTLA4-CD80/CD86 interactions play an important role in modulating the expression of BclXL.

To directly establish a functional link between ROS and T cell cycle progression and survival [42 ], T cells were activated with a stimulatory or a suppressive dose of Con A in the presence of different antioxidants (Fig. 4D) . GSH reduces increased oxidation in cells, exogenous catalase specifically cleaves excess H2O2 that diffuses out of the cell, and L-NMA is an inhibitor of nitric oxide (NO) synthase. In T cells activated with a stimulatory dose of Con A, these agents did not cause any significant effects on the proportion of cycling cells or T cell death across multiple experiments (Fig. 4E) . However, in T cells activated with a suppressive dose of Con A, these agents significantly increased T cell cycling and reduced the proportion of hypodiploid T cells (Fig. 4D and 4E) . These results clearly implicate H2O2 and NO in enhanced primary CD4+ T cell death on activation with a suppressive dose of Con A. Together, these results demonstrate that T cell death with a suppressive dose of Con A is dependent on ROS but is independent of Fas and caspase activity.

Functional roles of inhibitory cytokines, TGF-ß and IL-10, during Con A-mediated activation of CD4+ T cells
TGF-ß is known to decrease cell-cycle progression and enhance death in T cells, and there are several studies about the roles of CTLA4 ligation in TGF-ß production [23 24 25 ]. T cells activated with a suppressive or a stimulatory dose of Con A produced similar amounts of TGF-ß, and triggering with anti-CD28 did not modulate TGF-ß levels on activation with a suppressive dose of Con A (Table 1 ). However, on activation of T cells with a stimulatory dose of Con A and anti-CD28, TGF-ß levels were not detectable. This effect is similar to the effect of CD28 in greatly reducing the production of active TGF-ß on activation of T cells with P + I [13 ]. Increased levels of TGF-ß were produced by T cells activated with suppressive and stimulatory doses of Con A-mediated activation and CTLA4-CD80/CD86 blockade. In fact, CTLA4 blockade with a stimulatory dose of Con A resulted in over a 4.5-fold increase in active TGF-ß levels and appeared to be consistent with results obtained with respect to T cell cycling and survival (Figs. 1 and 3) . However, the ~2.7-fold increase in active TGF-ß observed in T cells activated with a suppressive dose of Con A and CTLA4-CD80/CD86 blockade was inconsistent with the results on T cell cycling and survival (Figs. 1 and 3) and led us to check for functional effects of TGF-ß in this system. Addition of a neutralizing antibody to TGF-ß1, the dominant isoform of TGF-ß in T cells, or fetuin, which binds to TGF-ß and sequesters it from TGF-ß receptors [45 ], did not display any significant effects on T cell activation with a stimulatory or a suppressive dose of Con A (Fig. 5A ). Although high levels of active TGF-ß are produced on Con A activation and CTLA4-CD80/C86 blockade, no functional role for TGF-ß was detected in this system. This is unlike the situation in P + I-mediated activation of T cells, where the addition of fetuin or anti-TGF-ß1 results in ~30% rescue of proliferation observed on CTLA4 blockade [13 ]. These results led us to study the functional role of another major inhibitory cytokine, IL-10 (Fig. 5B) . Dose-dependent rescue of inhibition of proliferation on CTLA4-CD80/CD86 blockade with a stimulatory dose of Con A was observed on neutralization using anti-IL-10, demonstrating that IL-10 played an important role. In T cells activated with a suppressive dose of Con A, a slight increase in the level of proliferation was observed with anti-IL10, but there was no fundamental change in the activation profile as that observed with a stimulatory dose of Con A and CTLA4-CD80/CD86 blockade. In summary, CTLA4-CD80/CD86 interactions, in suppressive and stimulatory-dose activation of T cells by Con A, inhibited TGF-ß production; however, TGF-ß does not play a functional role here. In fact, IL-10 is functionally more important during activation of T cells with a stimulatory dose of Con A and CTLA4-CD80/CD86 blockade.


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  		Table 1. Differences in Production of IL-2 and TGF-ß on Blockade of CTLA4-CD80/CD86 Interactions by CD4+ T Cells Activated with a Stimulatory or a Suppressive Dose of Con A



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  		Figure 5. IL-10 but not TGF-ß plays a major, functional role in CD4+ T cell activation with a stimulatory (1 µg/ml) dose of Con A and CTLA4-CD80/CD86 blockade. Cells were treated with anti-TGF-ß1 or fetuin (A) or with anti-IL-10 (B) in the presence or absence of blockade of CTLA4-CD80/CD86 interactions. Cells were activated for 36 h, followed by a 12-h pulse with 3H-thymidine.

Blockade of CTLA4-CD80/CD86 interactions on CD4+ T cells and activation with a suppressive dose of Con A results in largely IL-2-independent but CsA-sensitive proliferation
IL-2 is a key cytokine responsible for T cell proliferation. The amount of IL-2 secreted in culture supernatants was detected using a specific IL-2 ELISA. As seen in Table 1 , T cells activated with a stimulatory or a suppressive dose of Con A produced similar amounts of IL-2, and these were enhanced with anti-CD28. This was quite unlike the proliferation profile, where T cell activation with a stimulatory dose of Con A and control antibody or anti-CD28 displayed much higher proliferation compared with corresponding cultures treated with a suppressive dose of Con A (Fig. 1) . Conversely, anti-CTLA4 or mCTLA4hIgG1 treatment of CD4+ T cells decreased IL-2 production on activation with a stimulatory and a suppressive dose of Con A (Table 1) . Although the decreased IL-2 production is consistent with the inhibition of proliferation observed on CTLA4-CD80/CD86 blockade in T cells activated with a stimulatory dose of Con A, it is inconsistent with the enhanced proliferation observed on CTLA4-CD80/CD86 blockade and activation with a suppressive dose of Con A. These observations led us to study the functional role of IL-2 during activation of CD4+ T cells with different doses of Con A. The addition of exogenous IL-2 completely rescued the inhibition of proliferation observed with a stimulatory dose of Con A together with CTLA4-CD80/CD86 blockade (Fig. 6A ). This rescue in proliferation was also mediated with exogenous IL-4, although higher amounts were required compared with IL-2. It is most important that the addition of IL-2 or IL-4 did not result in any significant increase in proliferation of T cells activated with a suppressive dose of Con A. To further confirm the roles of IL-2 in this system, we used neutralizing antibodies against IL-2 (Fig. 6B) . Increased amounts of anti-IL-2 but not anti-IL-4 greatly reduced the proliferation of T cells activated with a stimulatory dose of Con A, demonstrating the key role of IL-2. However, only a mild reduction in proliferation was observed in T cells activated with a suppressive dose of Con A and blockade of CTLA4-CD80/CD86. Together, these results demonstrate that proliferation of T cells activated with a suppressive dose of Con A with CTLA4-B7 blockade was largely IL-2-independent, whereas T cell proliferation with a stimulatory dose of Con A was largely dependent on IL-2. Finally, we used CsA to determine the importance of the calcineurin pathway in this system (Fig. 6C) . The inhibition observed with CsA addition in Con A-mediated activation was comparable with that seen with plate-bound anti-CD3 (data not shown). Thus, T cells activated with Con A, in the absence or presence of CTLA4 blockade, used the CsA-sensitive calcineurin TCR signal transduction pathway involving nuclear factor activated T cell signaling to proliferate.



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  		Figure 6. Blockade of CTLA4-CD80/CD86 interactions on CD4+ T cells results in mainly IL-2-independent proliferation on activation with a suppressive (4 µg/ml) dose of Con A. Exogenous IL-2 or IL-4 (A) or neutralizing antibodies to IL-2 or IL-4 (B) in the indicated concentrations were added at 0 h to cells stimulated with a stimulatory or a suppressive dose of Con A in the presence of different antibodies. (C) CsA was added at the indicated concentrations at 0 h, and the inhibition of proliferation after activation for 36 h, followed by a 12-h pulse with 3H-thymidine, is depicted.


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DISCUSSION
 
Con A is a known T cell mitogen, but the mechanisms involved in this process are poorly studied. APCs are thought to be required for Con A-mediated T cell activation [35 , 37 ]. However, another study demonstrated that purified T cells are activated by Con A without the requirement for APCs [36 ]. We find that primary mouse CD4+ T cells expressed costimulatory receptors CD28 and CTLA4 and their ligands CD80 and CD86 after activation with Con A (Fig. 2) . These cells responded to Con A depending on the concentration used [38 ]: A low dose of Con A was stimulatory for CD4+ T cells, whereas a high dose of Con A suppressed proliferation followed by increased T cell death (Figs. 1 and 3) . To study the roles of costimulatory receptors CD28 and CTLA4 and their ligands CD80 and CD86 on CD4+ T cells activated with a stimulatory or a suppressive dose of Con A, three independent reagents were used. Anti-CD28 binds and signals via CD28, together with a stimulatory dose of Con A, to enhance T cell cytokine production and proliferation. It is interesting that anti-CD28 triggering, on activation with a suppressive dose of Con A, was not able to enhance cell survival, perhaps as a result of the dominant effects of the CTLA4-CD80/CD86 interactions in this system (Fig. 1) . It is also possible that CD28 signaling in the presence of a strong signal curtails T cell responses by inducing apoptosis [46 ]. CD28-CD80/CD86 interactions do not appear to be playing a major role, perhaps as a result of the fact that it does not bind effectively with hypoglycosylated B7 molecules present on mouse T cells [26 , 27 ]. As all our results are consistent with soluble anti-CTLA4 and mCTLA4hIgG1, most likely the major interactions observed are a result of CTLA4 binding to CD80/CD86. It is unlikely that CD80 and CD86 are signaling in this T cell:T cell system, as no major effects were observed on cross-linking with anti-CD80 and anti-CD86 (data not shown). It was shown previously that mCTLA4IgG1 does not reduce Con A binding to cell-surface molecules [37 ]. Consistent with this study, Con A binding to cell-surface molecules was not affected with anti-CTLA4 or mCTLA4IgG1, as preincubating activated cells with Con A did not decrease staining with FITC-conjugated anti-CTLA4, anti-CD80, or anti-CD86 (data not shown).

The roles of CTLA4-CD80/CD86 interactions post-activation with a stimulatory or a suppressive dose of Con A are summarized in Figure 7 . Binding of CTLA4 to CD80/CD86 on CD4+ T cells activated with a stimulatory dose of Con A-enhanced T cell cycling and survival, as blocking these interactions led to increased ROS and TGF-ß levels but decreased IL-2. This is similar to the roles of CTLA4-CD80/CD86 interactions on T cells activated with P + I [13 ]. It is important to point out that CD4+ T cell-proliferative responses to a stimulatory dose of Con A were reduced but were not abrogated completely (Fig. 1) on blocking CTLA4-CD80/CD86 interactions, suggesting roles for other cell-surface proteins in this process. Also, in T cells activated with a stimulatory dose of Con A and CTLA4-CD80/CD86 blockade, excess ROS was produced by 24 h (Fig. 3A and 3B) , followed by reduction in membrane potential (Fig. 3C and 3D) and reduced proliferation and cell cyling (Figs. 1C and 3F) by 36–48 h. A more dramatic phenotype was observed with T cells activated with a suppressive dose of Con A, which resulted in increased ROS production and greatly inhibited CD4+ T cell activation. Blocking CTLA4-CD80/CD86 responses under this condition led to greatly increased T cell cycling and survival, which was largely IL-2 independent. This pathway was sensitive to CsA, demonstrating dependence on the phosphatase calcineurin. IL-2-deficient T cells are activated with anti-CD3 and B7-transfected cells, demonstrating that IL-2 is not essential for B7-induced T cell proliferation [47 ]. Also, CD28-mediated proliferation of T cells involves IL-2-dependent and independent pathways [48 ]. It is most interesting that CD4+ T cell hyperproliferation in ctla4–/– mice is IL-2-independent [49 ]. Our results demonstrate that the roles of CTLA4-CD80/CD86 interactions differ based on the strength of activation of T cells. Thus, on activation with a suppressive dose of Con A, CTLA4-CD80/CD86 responses inhibit T cell responses, consistent with its role as a negative regulator during T cell activation [1 2 3 ]. These studies clearly demonstrate that CTLA4 is an "intelligent" costimulatory receptor that integrates signal strength information to modulate primary mouse CD4+ T cell activation.



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  		Figure 7. The functional effects of CTLA4-CD80/CD86 interactions in a CD4+ T cell:T cell activation model depend on the dose of Con A used for primary activation. These interactions are beneficial at stimulatory but are inhibitory at suppressive doses of Con A.

The role of CTLA4 in survival of T cells is controversial. CTLA4 ligation of activated T cells results in death [15 , 16 ] and enhances {gamma}-irradiation-induced apoptosis [17 ]. However, CTLA4 expression on differentiated T cells increases T cell survival in the AICD model [12 , 14 ]. In primary CD4+ T cells, activation by anti-CD3 and ligation of CTLA4 reduced T cell cycling without affecting survival [6 ]. However, in CD4+ T cells expressing a transgenic TCR, CTLA4 was found to reduce T cell expansion and survival [10 ]. Also, CD4+ T cells activated with P + I, together with blocking CTLA4-CD80/CD86 interactions, reduced T cell cycling and survival [13 ]. The role of CTLA4 in survival of primary T cells is unclear, and these discrepancies may be a result of the use of different populations of T cells (primary vs. differentiated) or T cells expressing TCRs with different affinities (anti-CD3 vs. a high-affinity, transgenic TCR) or a result of differences in activation conditions (P+I or anti-CD3). In this study, using primary CD4+ T cells, we clearly show that CTLA4-CD80/CD86 interactions modulate T cell cycling and survival depending on the dose of Con A. On activation with stimulatory doses of Con A, CTLA4-CD80/CD86 interactions enhance T cell survival (Fig. 3E and 3F) . Although the effects are much reduced, it is similar to the roles of these interactions observed in the P + I system [13 ]. However, the role of CTLA4-CD80/CD86 interactions in reducing T cell survival was uncovered on blocking CTLA4-CD80/CD86 interactions and activation of T cells with a suppressive dose of Con A. Notably, enhanced T cell death with suppressive doses of Con A is Fas- and caspase-independent (Fig. 4A and 4B) . Previously, Con A was shown to induce signaling ROS in mouse thymocytes, implicating flavonoid reduced nicotinamide adenine dinucleotide phosphate oxidase(s) but not NO synthase [39 ]. Here, we show that primary mouse CD4+ T cells activated with a suppressive dose of Con A induced excess ROS (Fig. 3A and 3B) , resulting in oxidative stress. Functionally, H2O2 and NO are involved in this process as exogenous catalase and L-NMA, independently or in combination, rescued primary CD4+ T cell cycling and survival on activation with a suppressive dose of Con A (Fig. 4E and 4F) . Together with the greatly modulated levels of BclXL and the functional roles of oxidative stress, it appears that enhanced primary mouse CD4+ T cell death with a suppressive dose of Con A is not activation-induced cell death but most likely autonomous T cell death [42 ].

Although many studies have demonstrated that CTLA4-CD80/CD86 interactions result in increased TGF-ß production, the functional roles of CTLA4-modulated TGF-ß levels are controversial [23 24 25 ]. Unlike other reports where CTLA4-mediated production of T cell inhibitory cytokines was limited to specific subsets of regulatory T (Treg) cells, our observations are likely to be a property of the global CD4+ T cell population, as depletion of CD25+CD4+ Treg cells prior to culture did not have any effects on the activation profile on blockade (data not shown). Blockade of CTLA4-CD80/CD86 interactions enhanced TGF-ß production by CD4+ T cells activated with P + I. The TGF-ß produced in this system did play a functional role by enhancing T cell death [13 ]. In the current study, anti-CD28 triggering greatly reduced the amount of TGF-ß produced by T cells stimulated with Con A. However, blockade of CTLA4-CD80/CD86 interactions also enhanced active TGF-ß production in T cells activated with a stimulatory and a suppressive dose of Con A; however, no significant rescue was observed with fetuin or specific anti-TGF-ß1 antibodies. The reasons for the lack of a functional effect of TGF-ß are unclear and led us to enquire about the role of another immunosuppressive cytokine, IL-10. A recent study demonstrated that CTLA4 ligation reduces interferon-{gamma} secretion by T cells in an IL-10-dependent manner. Also, CTLA4-induced IL-10 may play an important role in anti-tumor T cell responses [50 ]. Although no major effect on neutralizing of IL-10 was observed with a suppressive dose of Con A, significant rescue in proliferation was observed in T cells activated with a stimulatory dose of Con A, together with blockade of CTLA4-CD80/CD86 interactions (Fig. 5B) . Thus, IL-10 is the key suppressive cytokine and is likely to be a major player in the cell-cycle arrest observed in this system. This is unlike the P + I-mediated activation system [13 ], where IL-10 does not play a role (data not shown). As CD4+ T cells are known to express different cytokines depending on varying doses of antigen [51 ], it is possible that expression of these immunosuppressive cytokines may differ depending on the primary activation conditions. Further studies are required to address the expression, cross-talk, and functional roles of TGF-ß and IL-10 on T cells activated under different conditions.

There are at least three studies that demonstrate a role for CTLA4 in modulating immune responses depending on the strength of signal. First, higher levels of CTLA4 accumulation at the immunological synapse are found with increased signal strength. Thus, a strong signal results in increased CTLA4 surface expression, which inhibits T cell activation [52 ]. Second, stimulation of T cells with high concentrations of antigen, together with CTLA4 blockade, favors a T helper cell type 2 response [32 ]. Finally, a role for CTLA4 in enhancing T cell responses was demonstrated in a study using an autoimmune encephalitis model. Here, immunization with a disease antagonistic peptide, but not a disease agonistic peptide, together with CTLA4 blockade inhibit generation of cross-reactive T cell clones. It is possible that antagonistic peptides generate sub-optimal primary signals, and CTLA4 interactions enhance T cell responses under this condition [33 ]. It is most important that CTLA4 blockade inhibits or enhances the generation of T cells expressing distinct TCRs of identical specificities [32 ]. Our results about the roles of CTLA4-CD80/CD86 interactions in modulating T cell activation, based on the dose of Con A used for activation, are consistent with these studies. However, validation of this model in other T cell activation systems is required. This point is particularly relevant for studies on T cell activation, as Con A binds to additional glycoprotein surface receptors in addition to the TCR [53 ]; hence, the functional consequences of activating T cells with Con A or anti-CD3 may be different [41 ]. We find that increasing ionomycin concentration, but not PMA, on activation of CD4+ T cells with P + I [13 ] abrogates T cell cycle arrest and death on blocking CTLA4-CD80/CD86 interactions by inducing high levels of IL-2 (data not shown). Preliminary experiments (data not shown) suggest that CTLA4-CD80/CD86 interactions enhance CD4+ T cell activation after activation with soluble anti-CD3 cross-linking, whereas these act in an inhibitory manner in the presence of plate-bound anti-CD3, which sends a stronger signal [54 ].

Two mutually nonexclusive models have been proposed to explain the role of CTLA4 during immune responses, threshold, and attenuator [2 ]. The former predicts that CTLA4 sets a stimulatory threshold for optimal T cell activation. Thus, in the absence of CTLA4 or with CTLA4 blockade, T cells proliferate in response to weak activation signals (e.g., TCR-MHC signals during peripheral survival of CD4+ T cells). Conversely, in the presence of a strong signal (e.g., inflammation by pathogens), CTLA4 regulates the extent of T cell division after initial activation. In this case, in the presence of CTLA4 blockade or ctla4–/–, cells divide with greater frequency. Both these models, with some modifications, appear to be at play in this study. It is possible that with a stimulatory dose of Con A, the threshold model plays an important role, as CTLA4 integrates the signal strength to enhance T cell activation. Conversely, it is possible that on T cell activation with suppressive doses of Con A, the attenuator model comes into play. Therefore, blocking CTLA4-CD80/CD86 interactions reduces signal strength to enhance T cell cycling and survival. Based on these results, we suggest that CTLA4-CD80/CD86 interactions on CD4+ T cells enhance T cell activation in the presence of a suboptimal or stimulatory signal, whereas CTLA4-CD80/CD86 interactions inhibit T cell activation in the presence of a strong or overly strong signal. It is possible that CTLA4-CD80/CD86 interactions inhibit the generation of dominant TCRs, which recognize antigens with a high affinity, whereas the same interactions may enhance the proliferation of T cells with lower affinities to enlarge the T cell immune response. These results are consistent with studies that demonstrate CTLA4-CD80/CD86 interactions regulate the diversity and the extent of the primary CD4+ T cell immune response [2 , 32 , 33 , 52 ]. In summary, this study clearly demonstrates that CTLA4-CD80/CD86 interactions on primary mouse CD4+ T cells modulate T cell activation depending on the dose of Con A. This data implicate the strength of primary signal in conjunction with CTLA4-CD80/CD86 interactions to modulate primary T cell responses. Further studies are required to understand the mechanisms by which CTLA4, a single receptor, can switch from an enhancer to a negative modulator of CD4+ T cell cycling and survival, depending on the signal strength used for activation.


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ACKNOWLEDGEMENTS
 
This study was supported by a grant from the Department of Biotechnology, Government of India. S. M. was awarded a research fellowship from CSIR. We are grateful to Dr. S. Rath for insightful comments about this study. Dr. P. Kondaiah provided the CCL64 cell line for the TGF-ß bioassay and neutralizing antibodies to TGF-ß. We thank Prof. T. Ramasarma for suggestions on oxidative stress, Dr. A. Sarin for caspase-inhibitors, and Drs. V. Bal and S. Rath for access to lpr–/– mice. The assistance of Dr. O. Joy and H. Krishnan, DBT-FACS facility, is highly appreciated.

Received November 5, 2004; revised January 20, 2005; accepted February 22, 2005.


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